Characteristics of drug resistance mutations in ART-experienced HIV-1 patients with low-level viremia in Zhengzhou City, China

Although most people living with HIV (PLWH) receiving antiretroviral therapy (ART) achieve continuous viral suppression, some show detectable HIV RNA as low-level viremia (LLV) (50–999 copies/mL). Drug resistance mutations (DRMs) in PLWH with LLV is of particular concern as which may lead to treatment failure. In this study, we investigated the prevalence of LLV and LLV-associated DRMs in PLWH in Zhengzhou City, China. Of 3616 ART-experienced PLWH in a long-term follow-up cohort from Jan 2022 to Aug 2023, 120 were identified as having LLV. Of these PLWH with LLV, we obtained partial pol and integrase sequences from 104 (70 from HIV-1 RNA and 34 from proviral DNA) individuals. DRMs were identified in 44 individuals. Subtyping analysis indicated that the top three subtypes were B (48.08%, 50/104), CRF07_BC (31.73%, 33/104), and CRF01_AE (15.38%, 16/104). The proportions of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs) associated DRMs were 23.83% (24/104), 35.58% (37/104), 5.77% (6/104), and 3.85% (4/104), respectively, which contributed to an overall prevalence of 42.31% (44/104). When analyzed by individual DRMs, the most common mutation(s) were V184 (18.27%, 19/104), followed by V179 (11.54%, 12/104), K103 (9.62%, 10/104), Y181 (9.62%, 10/104), M41 (7.69%, 8/104), and K65R (7.69%, 8/104). The prevalence of DRMs in ART-experienced PLWH with LLV is high in Zhengzhou City and continuous surveillance can facilitate early intervention and provision of effective treatment.

We further analyzed each drug independently.NVP dominated in high-level, DOR and RPV dominated in medium-level, ABC dominated in low-level, and ETR dominated in potentially low-level of resistant drugs.As more than 90% of PLWH would choose the current free drug regimens for treatment, prevalence of DRMs against the available free ART drugs (ABC, AZT, 3TC and TDF of NRTIs, NVP and EFV of NNRTIs, and LPV/r of PIs) in China were analyzed.Our results indicated, the overall prevalence of DRMs to the seven free ART drugs was 36.54%(38/104), while DRMs that led to low-or higher-level resistance was 25.96% (27/104).These included low-or higher-level resistance to ABC, AZT, 3TC and TDF of the NRTIs in 21.15% (22/104), 7.69% (8/104), 21.15% (22/104), and 15.38% (16/104), respectively; to NVP and EFV of the NNRTIs in 24.04% (25/104) and 24.04% (25/104), respectively; and to LPV/r of the PIs in 2.88% (3/104).Detailed information about the DRMsrelated drug resistance to ART regimens is shown in Fig. 3.

Influencing factors of DRMs in LLV individuals
Univariate logistic regression analysis was used to analyze the influencing factors associated with DRMs in PLWH with LLV (Tables 3 and 4).According to our results, individuals of less than 30-years old were more likely to develop DRMs than other age groups (OR = 22.286, 95% CI = 2.379-208.789,P < 0.05).The genotyping success rates of PLWH with VL of 50-200, 201-400, 401-999 copies/mL groups were 85.14% (63/74), 84.21% (16/19), and 81.82% (9/11), respectively, and there was no statistical difference in genotyping success rate (data not shown).The incidence of DRMs in LLV individuals with VL between 401 and 999 copies/mL (OR = 9.375, 95% CI = 1.878-46.790,P < 0.05) and 201 and 400 copies/mL (OR = 2.865, 95% CI = 1.020-8.045,P < 0.05) was significantly higher than that between 50 and 200 copies/mL.The association analysis indicated that individuals with LLV receiving 2NRTIs + PI/r regimen when LLV (including both iLLV and pLLV) was detected were more likely to develop DRMs (OR = 19.500,95% CI = 3.585-106.077,P < 0.05)..Notably, due to historical reasons in the 1990s and a large proportion of such patients, plasmapheresis accounted for 17.31% (18/104) of total LLV individuals.Our data showed that the incidence of DRM increases with the increase of VL in LLV individuals, especially in pLLV individuals (Tables 3 and 4).Both iLLV and pLLV during ART have been reported to be associated with increased risk of subsequent virologic failure 13 .A study in Botswana indicated that a single LLV during ART can strongly predict the risk of future virologic failure 23 .A study in the US revealed that the magnitude of LLV was the primary driver of evolution rate at both DRM and non-DRM sites, and higher VL was associated with the development of DRM 24 .These findings may provide applicable insights to the management of LLV individuals during ART.
Our results are consistent with reports in that LLV individuals using PI-based regimens for initial ART are less likely to develop DRMs than those using NNRTI-based regimens, while individuals with the VL between 401 and 999 copies /mL (OR = 9.375, 95% CI = 1.878-46.790,P < 0.05) and 201 and 400 copies /mL (OR = 2.865, 95% CI = 1.020-8.045,P < 0.05) were more likely to develop DRMs than that between 50 and 200 copies/mL 16,28 .It is worth noting that DRMs are more likely to be detected in individuals receiving PI-based regimens when LLV is detected (OR = 19.500,95% CI = 3.585-106.077,P < 0.05), which most possibly is due to previous failure to first-line antiretroviral regimens consisting of NNRTIs and NRTIs as PIs-associated DRMs are rarely detected.
Our study has several limitations.First, the small sample size could have limited the power of the statistical and correlation analysis.Second, individuals in the VL group of 50-200 copies/mL accounted for 71.15%, which was significantly higher, and may bring bias to subsequent analysis.Third, influencing factors could not be accurately assessed for the lack of some clinical information in some individuals, such as occupation, education, CD4 + T cell count, and adherence.Fourth, some of the results were interpreted using proviral DNA-based genotyping, concordance between proviral DNA and RNA genotyping needs to be further improved.Nevertheless,

Ethical statement
This study was approved by the Institutional Ethics Committee of The Sixth People's Hospital of Zhengzhou, China (IEC-KY-2022-005-2) and performed in compliance with all relevant ethical regulations such as the Declaration of Helsinki (2008).Signed informed consent was obtained from each individual before the collection of blood samples.

Study population
There are approximately 4000 PLWH in Zhengzhou City and each year they come to the Sixth People's Hospital of Zhengzhou for annual monitoring of their VL and CD4+ T-cell count.PLWH who had experienced ART for at least 6 months when visiting the Sixth People's Hospital of Zhengzhou from January 2022 to August 2023 and exhibited a VL greater than 50 copies/mL and less than 1,000 copies/mL at one time point (iLLV/blips) and/ or two consecutive time points (pLLV) with previously undetected VL (< 50 copies/mL) were included in this

Nucleic acid purification
DNA extraction: whole blood samples were collected and centrifuged at low speed to obtain the buffy coat, which were then used for HIV-1 DNA extraction following the instructions of a Blood Genomic DNA Extraction kit (CWBio, Jiangsu, China) as per the manufacturer's instructions.RNA extraction: plasma samples (1-5 mL) were concentrated at 28,000 g for 30 min at 4 °C by ultracentrifugation to enrich HIV.The pellet was resuspended in phosphate buffered saline (0.01 M, pH 7.2) and then used

Genotypic drug resistance testing
An In-house genotypic drug resistance testing was performed as described previously 27 .Briefly, HIV-1 RNA was used to amplify HIV-1 partial pol gene sequence and full-length integrase (INT) gene sequence using reverse transcription and nested-PCR, or alternatively HIV proviral DNA was used when HIV RNA based amplification failed.The HIV-1 pol and INT gene was reverse transcribed using the specific primer R5073 and a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA).The target sequence of pol gene (approximately 1300 bp) was amplified by nested PCR using LA Taq™ Version 2.0 (Takara, Shiga, Japan) with two primer sets (PRO-F1 and RT-R1 for first-round PCR, and PRO-F2 and RT-R2 for second-round PCR).Each amplified fragment was sequenced with two primer sets (CF1, CF3, CR2, and CR4 as the primary sequencing primer set; CF2, CF4, CR1, and CR3 as the backup sequencing primer set) (Supplementary Table 1).The target sequence of INT gene (approximately 700 bp) was amplified by nested PCR using two primer sets (F4181 and R5073 for first-round PCR, and F4379 and R5057 for second-round PCR).Each amplified fragment was sequenced using two primer sets (CF5 and CR5 as the primary sequencing primer set, and CF6 and R5057 as backup sequencing primer set) (Supplementary Table 1).The positive PCR products were purified, sequenced using Sanger sequencing, and then submitted to the regularly updated Stanford HIV-1 drug resistance database (http:// hivdb.stanf ord.edu/) for DRMs and antiretroviral susceptibility analysis.Polymorphic mutations and polymorphic accessory mutations in combination with other DRMs might contribute to reduced susceptibility of certain antiretroviral drugs, and they are thus included in our analysis.

Statistical analysis
Statistical analysis was carried out using the SPSS statistics program for Windows (IBM SPSS Statistics, version 21).Continuous variables were expressed as mean ± standard deviation or a median with its interquantile range (IQR).Categorical variables were expressed as numbers or percentages.The differences between or among groups were analyzed by Student's t-test or Chi-squared test; a P < 0.05 was considered to be statistically significant.

Table 1 .
21aracteristicsDiscussionIn this study we investigated the prevalence of, and DRMs associated with LLV in PLWH in Zhengzhou City.Due to technical problems, most RNA-based genotyping assays require the HIV VL to be above 1000 copies/ mL, which is also an important reason for the limited data on DRMs in LLV individuals.Therefore, although genotypic drug resistance assay based on HIV proviral DNA is not routinely used for clinical monitoring, it still can be very useful in PLWH when plasma sequencing is not successful21.Our results showed that among 3616 ART-experienced PLWH in a long-term follow-up cohort from Jan 2022 to Aug 2023 in Zhengzhou City, 120 were identified as having LLV, giving a prevalence of 3.32%.Of these LLV individuals, males accounted for 78.10% of people living with HIV (PLWH) who presented with low-level viremia in Zhengzhou City, 2023.Data are presented as n (%) or median (IQR); IQR, interquartile range; significance for differences was measured using Chi-squared test, Fisher's Exact test, or Kruskal-Wallis test.HSX, heterosexual orientation; MSM, men who have sex with men; MTCT, mother-to-child transmition; PL, plasmapheresis; IDU, injection drug use; OTH, others, including patients whose risk factors were unknown or patients who did not provide information; TDRM, transmitted drug-resistance mutations; NRTI, nucleotide reverse transcriptase inhibitors; NNRTI, non-nucleoside reverse transcriptase inhibitors; PI, protease inhibitors; INSTI, integrase strand transfer inhibitors.Vol.:(0123456789) Scientific Reports | (2024) 14:10620 | https://doi.org/10.1038/s41598-024-60965-zwww.nature.com/scientificreports/(82/104).When stratified by age, LLV incidence among PLWH was higher in the age group of 30-39 (25.96%, 27/104) and 50-59 (25.00%, 26/104), and the incidence of PLWH over 50-year old was 44.23% (46/104), which is consistent with other studies in China

Table 2 .
Distribution and prevalence of DRMs among HIV-1 subtypes in PLWH with LLV in Zhengzhou City, 2023.Univariate logistic regression analysis was performed.CRF, circulating recombinant forms; DRMs, drug resistance mutations.

Table 3 .
Analysis of influencing factors of DRMs among PLWH with LLV in Zhengzhou City, 2023.

Table 4 .
Analysis of influencing factors of DRMs among PLWH between iLLV and pLLV in Zhengzhou City, 2023.RNA extraction using an RNA extraction kit (Liferiver, Shanghai, China) by following the manufacturer's instructions.